Previous studies in this laboratory have been concerned with the various parameters of the regulation of carotene synthesis by light. It is now planned to investigate biosynthetic pathway of cyclic carotenes using soluble enzyme preparations. Enzyme systems capable of converting various precursors into beta-carotene will be prepared by disrupting the cells ultrasonically and separatng by centrifugation particles containing carotene-protein complexes which are localized in the cell envelope. Protein fraction will be separated by grinding the particles with glass beads and by preparing an acetone powder. Further purification will be carried out by extracting the ground material and the acetone powder with phosphate buffer and fractionation with ammonium sulfate. Carotenogenic enzyme activities will be assayed at each step of purification by using (C-14)-lycopene and other acyclic carotenes as substrates. The kinetics and the cofactor requirements of the reactions will be determined. Once the soluble enzyme preparations are available, studies will be undertaken to confirm the in vivo observations made in this laboratory that nicotine inhibits the cyclization of lycopene to form beta-carotene. The mode of nicotine inhibition will be determined from the kinetic data. In addition to nicotine, 2-(4-chlorophenylthio)- thiethylamine hydrochloride (CPTA) has also been reported to stimulate the accumulation of lycopene rather than beta-carotene. Effectiveness of this compound as an inhibitor of the cyclization reaction(s) will be determined. Such a study should reveal the comparative mode of action of CPTA and nicotine. From the long-range point of view, the preparation of carotenogenic enzyme systems is essential for investigating the mode of regulation of carotene synthesis by light and antimycin A in Mycobacterium marinum.